Secreted proteins and polynucleotides encoding them

ABSTRACT

Novel polynucleotides and the proteins encoded thereby are disclosed.

This application is a continuation-in-part of application Ser. No.08/686,878, filed Jul. 26, 1996.

FIELD OF THE INVENTION

The present invention provides novel polynucleotides and proteinsencoded by such polynucleotides, along with therapeutic, diagnostic andresearch utilities for these polynucleotides and proteins.

BACKGROUND OF THE INVENTION

Technology aimed at the discovery of protein factors (including e.g.,cytokines, such as lymphokines, interferons, CSFs and interleukins) hasmatured rapidly over the past decade.

The now routine hybridization cloning and expression cloning techniquesclone novel polynucleotides "directly" in the sense that they rely oninformation directly related to the discovered protein (i.e., partialDNA/amino acid sequence of the protein in the case of hybridizationcloning; activity of the protein in the case of expression cloning).More recent "indirect" cloning techniques such as signal sequencecloning, which isolates DNA sequences based on the presence of a nowwell-recognized secretory leader sequence motif, as well as variousPCR-based or low stringency hybridization cloning techniques, haveadvanced the state of the art by making available large numbers ofDNA/amino acid sequences for proteins that are known to have biologicalactivity by virtue of their secreted nature in the case of leadersequence cloning, or by virtue of the cell or tissue source in the caseof PCR-based techniques. It is to these proteins and the polynucleotidesencoding them that the present invention is directed.

SUMMARY OF THE INVENTION

In one embodiment, the present invention provides a compositioncomprising an isolated polynucleotide selected from the group consistingof:

(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:2;

(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:2from nucleotide 342 to nucleotide 542;

(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:2from nucleotide 402 to nucleotide 542;

(d) a polynucleotide comprising the nucleotide sequence of the fulllength protein coding sequence of clone AJ172₋₋ 2 deposited underaccession number ATCC 98115;

(e) a polynucleotide encoding the full length protein encoded by thecDNA insert of clone AJ172₋₋ 2 deposited under accession number ATCC98115;

(f) a polynucleotide comprising the nucleotide sequence of the matureprotein coding sequence of clone AJ172₋₋ 2 deposited under accessionnumber ATCC 98115;

(g) a polynucleotide encoding the mature protein encoded by the cDNAinsert of clone AJ172₋₋ 2 deposited under accession number ATCC 98115;

(h) a polynucleotide encoding a protein comprising the amino acidsequence of SEQ ID NO:3;

(i) a polynucleotide encoding a protein comprising a fragment of theamino acid sequence of SEQ ID NO:3 having biological activity;

(j) a polynucleotide which is an allelic variant of a polynucleotide of(a)-(g) above;

(k) a polynucleotide which encodes a species homologue of the protein of(h) or (i) above.

Preferably, such polynucleotide comprises the nucleotide sequence of SEQID NO:2 from nucleotide 342 to nucleotide 542; the nucleotide sequenceof SEQ ID NO:2 from nucleotide 402 to nucleotide 542; the nucleotidesequence of the full length protein coding sequence of clone AJ172₋₋ 2deposited under accession number ATCC 98115; or the nucleotide sequenceof the mature protein coding sequence of clone AJ172₋₋ 2 deposited underaccession number ATCC 98115. In other preferred embodiments, thepolynucleotide encodes the full length or mature protein encoded by thecDNA insert of clone AJ172₋₋ 2 deposited under accession number ATCC98115.

Other embodiments provide the gene corresponding to the cDNA sequence ofSEQ ID NO:2, SEQ ID NO:1 or SEQ ID NO:4.

In other embodiments, the present invention provides a compositioncomprising a protein, wherein said protein comprises an amino acidsequence selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO:3;

(b) fragments of the amino acid sequence of SEQ ID NO:3; and

(c) the amino acid sequence encoded by the cDNA insert of clone AJ172₋₋2 deposited under accession number ATCC 98115; the protein beingsubstantially free from other mammalian proteins. Preferably suchprotein comprises the amino acid sequence of SEQ ID NO:3.

In certain preferred embodiments, the polynucleotide is operably linkedto an expression control sequence. The invention also provides a hostcell, including bacterial, yeast, insect and mammalian cells,transformed with such polynucleotide compositions.

Processes are also provided for producing a protein, which comprise:

(a) growing a culture of the host cell transformed with suchpolynucleotide compositions in a suitable culture medium; and

(b) purifying the protein from the culture.

The protein produced according to such methods is also provided by thepresent invention. Preferred embodiments include those in which theprotein produced by such process is a mature form of the protein.

Protein compositions of the present invention may further comprise apharmaceutically acceptable carrier. Compositions comprising an antibodywhich specifically reacts with such protein are also provided by thepresent invention.

Methods are also provided for preventing, treating or ameliorating amedical condition which comprises administering to a mammalian subject atherapeutically effective amount of a composition comprising a proteinof the present invention and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are schematic representations of the pED6 and pNotSvectors used for deposit of clones disclosed herein.

DETAILED DESCRIPTION

Isolated Proteins and Polynucleotides

Nucleotide and amino acid sequences are reported below for each cloneand protein disclosed in the present application. In some instances thesequences are preliminary and may include some incorrect or ambiguousbases or amino acids. The actual nucleotide sequence of each clone canreadily be determined by sequencing of the deposited clone in accordancewith known methods. The predicted amino acid sequence (both full lengthand mature) can then be determined from such nucleotide sequence. Theamino acid sequence of the protein encoded by a particular clone canalso be determined by expression of the clone in a suitable host cell,collecting the protein and determining its sequence.

For each disclosed protein applicants have identified what they havedetermined to be the reading frame best identifiable with sequenceinformation available at the time of filing. Because of the partialambiguity in reported sequence information, reported protein sequencesinclude "Xaa" designators. These "Xaa" designators indicate either (1) aresidue which cannot be identified because of nucleotide sequenceambiguity or (2) a stop codon in the determined nucleotide sequencewhere applicants believe one should not exist (if the nucleotidesequence were determined more accurately).

As used herein a "secreted" protein is one which, when expressed in asuitable host cell, is transported across or through a membrane,including transport as a result of signal sequences in its amino acidsequence. "Secreted" proteins include without limitation proteinssecreted wholly (e.g., soluble proteins) or partially (e.g., receptors)from the cell in which they are expressed. "Secreted" proteins alsoinclude without limitation proteins which are transported across themembrane of the endoplpasmic reticulum.

Clone "AJ172 2"

A polynucleotide of the present invention has been identified as clone"AJ172₋₋ 2". AJ172₋₋ 2 was isolated from a human adult testes cDNAlibrary using methods which are selective for cDNAs encoding secretedproteins. AJ172₋₋ 2 is a full-length clone, including the entire codingsequence of a secreted protein (also referred to herein as "AJ172₋₋ 2protein").

The nucleotide sequence of the 5' portion of AJ172₋₋ 2 as presentlydetermined is reported in SEQ ID NO:1. An additional internal nucleotidesequence from AJ172₋₋ 2 as presently determined is reported in SEQ IDNO:2. What applicants believe is the proper reading frame and thepredicted amino acid sequence encoded by such internal sequence isreported in SEQ ID NO:3. Amino acids 1 to 20 of SEQ ID NO:3 are apredicted leader/signal sequence, with the predicted mature amino acidsequence beginning at amino acid 21. Additional nucleotide sequence fromthe 3' portion of AJ172₋₋ 2, including the polyA tail, is reported inSEQ ID NO:4.

The EcoRI/NotI restriction fragment obtainable from the depositcontaining clone AJ172₋₋ 2 should be approximately 2700 bp.

The nucleotide sequence disclosed herein for AJ172₋₋ 2 was searchedagainst the GenBank database using BLASTA/BLASTX and FASTA searchprotocols. AJ172₋₋ 2 demonstrated at least some homology with thefollowing sequences: Friend murine leukemiz virus (M93134, BlastX);Moloney murine leukemia virus genome (J02255, BlastN); pol protein,Gibbon leukemia virus (M26927, BlastX); and and EST identified as"yh46a09.s1 Homo sapiens cDNA clone 132760 3'" (R27389, BlastN). Basedupon homology, AJ172₋₋ 2 proteins and each homologous protein or peptidemay share at least some activity.

Deposit of Clones

Clone AJ172₋₋ 2 was deposited on Jul. 25, 1996 with the American TypeCulture Collection under accession number ATCC 98115, from which theclone comprising a particular polynucleotide is obtainable. This is acomposite deposit comprising a mixture of cells containing AJ172₋₋ 2with cell containing other clones. Each clone has been transfected intoseparate bacterial cells (E. coli) in this composite deposit.

Each clone can be removed from the vector in which it was deposited byperforming an EcoRI/NotI digestion (5' cite, EcoRI; 3' cite, NotI) toproduce the appropriately sized fragment for such clone (approximateclone size fragment are identified below). Each clone was deposited ineither the pED6 or pNotS vector depicted in FIG. 1A or FIG. 1B. In someinstances, the deposited clone can become "flipped" (i.e., in thereverse orientation) in the deposited isolate. In such instances, thecDNA insert can still be isolated by digestion with EcoRI and NotI.However, NotI will then produce the 5' cite and EcoRI will produce the3' cite for placement of the cDNA in proper orientation for expressionin a suitable vector. The cDNA may also be expressed from the vectors inwhich they were deposited.

Bacterial cells containing a particular clone can be obtained from thecomposite deposit as follows:

An oligonucleotide probe or probes should be designed to the sequencethat is known for that particular clone. This sequence can be derivedfrom the sequences provided herein, or from a combination of thosesequences. The sequence of the oligonucleotide probe that was used toisolate each full-length clone is identified below, and should be mostreliable in isolating the clone of interest.

    ______________________________________                                        Clone              Probe Sequence                                             ______________________________________                                        AJ172.sub.-- 2     SEQ ID NO:5                                                ______________________________________                                    

In the sequences listed above which include an N at position 2, thatposition is occupied in preferred probes/primers by a biotinylatedphosphoaramidite residue rather than a nucleotide (such as, for example,that produced by use of biotin phosphoramidite(1-dimethoxytrityloxy-2-(N-biotinyl-4-aminobutyl)-propyl-3-O-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramadite)(Glen Research, cat. no. 10-1953)).

The design of the oligonucleotide probe should preferably follow theseparameters:

(a) It should be designed to an area of the sequence which has thefewest ambiguous bases ("N's"), if any;

(b) It should be designed to have a T_(m) of approx. 80° C. (assuming 2°for each A or T and 4 degrees for each G or C).

The oligonucleotide should preferably be labeled with g-³² P ATP(specific activity 6000 Ci/mmole) and T4 polynucleotide kinase usingcommonly employed techniques for labeling oligonucleotides. Otherlabeling techniques can also be used. Unincorporated label shouldpreferably be removed by gel filtration chromatography or otherestablished methods. The amount of radioactivity incorporated into theprobe should be quantitated by measurement in a scintillation counter.Preferably, specific activity of the resulting probe should beapproximately 4e+6 dpm/pmole.

The bacterial culture containing the pool of full-length clones shouldpreferably be thawed and 100 μl of the stock used to inoculate a sterileculture flask containing 25 ml of sterile L-broth containing ampicillinat 100 μg/ml. The culture should preferably be grown to saturation at37° C., and the saturated culture should preferably be diluted in freshL-broth. Aliquots of these dilutions should preferably be plated todetermine the dilution and volume which will yield approximately 5000distinct and well-separated colonies on solid bacteriological mediacontaining L-broth containing ampicillin at 100 μg/ml and agar at 1.5%in a 150 mm petri dish when grown overnight at 37° C. Other knownmethods of obtaining distinct, well-separated colonies can also beemployed.

Standard colony hybridization procedures should then be used to transferthe colonies to nitrocellulose filters and lyse, denature and bake them.

The filter is then preferably incubated at 65° C. for 1 hour with gentleagitation in 6×SSC (20×stock is 175.3 g NaCl/liter, 88.2 g Nacitrate/liter, adjusted to pH 7.0 with NaOH) containing 0.5% SDS, 100μg/ml of yeast RNA, and 10 mM EDTA (approximately 10 mL per 150 mmfilter). Preferably, the probe is then added to the hybridization mix ata concentration greater than or equal to 1e+6 dpm/mL. The filter is thenpreferably incubated at 65° C. with gentle agitation overnight. Thefilter is then preferably washed in 500 mL of 2×SSC/0.5% SDS at roomtemperature without agitation, preferably followed by 500 mL of2×SSC/0.1% SDS at room temperature with gentle shaking for 15 minutes. Athird wash with 0.1×SSC/0.5% SDS at 65° C. for 30 minutes to 1 hour isoptional. The filter is then preferably dried and subjected toautoradiography for sufficient time to visualize the positives on theX-ray film. Other known hybridization methods can also be employed.

The positive colonies are picked, grown in culture, and plasmid DNAisolated using standard procedures. The clones can then be verified byrestriction analysis, hybridization analysis, or DNA sequencing.

Fragments of the proteins of the present invention which are capable ofexhibiting biological activity are also encompassed by the presentinvention. Fragments of the protein may be in linear form or they may becyclized using known methods, for example, as described in H. U.Saragovi, et al., Bio/Technology 10, 773-778 (1992) and in R. S.McDowell, et al., J. Amer. Chem. Soc. 114, 9245-9253 (1992), both ofwhich are incorporated herein by reference. Such fragments may be fusedto carrier molecules such as immunoglobulins for many purposes,including increasing the valency of protein binding sites. For example,fragments of the protein may be fused through "linker" sequences to theFc portion of an immunoglobulin. For a bivalent form of the protein,such a fusion could be to the Fc portion of an IgG molecule. Otherimmunoglobulin isotypes may also be used to generate such fusions. Forexample, a protein--IgM fusion would generate a decavalent form of theprotein of the invention.

The present invention also provides both full-length and mature forms ofthe disclosed proteins. The full-length form of the such proteins isidentified in the sequence listing by translation of the nucleotidesequence of each disclosed clone. The mature form of such protein may beobtained by expression of the disclosed full-length polynucleotide(preferably those deposited with ATCC) in a suitable mammalian cell orother host cell. The sequence of the mature form of the protein may alsobe determinable from the amino acid sequence of the full-length form.

The present invention also provides genes corresponding to the cDNAsequences disclosed herein. The corresponding genes can be isolated inaccordance with known methods using the sequence information disclosedherein. Such methods include the preparation of probes or primers fromthe disclosed sequence information for identification and/oramplification of genes in appropriate genomic libraries or other sourcesof genomic materials.

Where the protein of the present invention is membrane-bound (e.g., is areceptor), the present invention also provides for soluble forms of suchprotein. In such forms part or all of the intracellular andtransmembrane domains of the protein are deleted such that the proteinis fully secreted from the cell in which it is expressed. Theintracellular and transmembrane domains of proteins of the invention canbe identified in accordance with known techniques for determination ofsuch domains from sequence information.

Species homologs of the disclosed polynucleotides and proteins are alsoprovided by the present invention. Species homologs may be isolated andidentified by making suitable probes or primers from the sequencesprovided herein and screening a suitable nucleic acid source from thedesired species.

The invention also encompasses allelic variants of the disclosedpolynucleotides or proteins; that is, naturally-occurring alternativeforms of the isolated polynucleotide which also encode proteins whichare identical, homologous or related to that encoded by thepolynucleotides.

The isolated polynucleotide of the invention may be operably linked toan expression control sequence such as the pMT2 or pED expressionvectors disclosed in Kaufman et al., Nucleic Acids Res. 19, 4485-4490(1991), in order to produce the protein recombinantly. Many suitableexpression control sequences are known in the art. General methods ofexpressing recombinant proteins are also known and are exemplified in R.Kaufman, Methods in Enzymology 185, 537-566 (1990). As defined herein"operably linked" means that the isolated polynucleotide of theinvention and an expression control sequence are situated within avector or cell in such a way that the protein is expressed by a hostcell which has been transformed (transfected) with the ligatedpolynucleotide/expression control sequence.

A number of types of cells may act as suitable host cells for expressionof the protein. Mammalian host cells include, for example, monkey COScells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, humanepidermal A431 cells, human Colo2O5 cells, 3T3 cells, CV-1 cells, othertransformed primate cell lines, normal diploid cells, cell strainsderived from in vitro culture of primary tissue, primary explants, HeLacells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells.

Alternatively, it may be possible to produce the protein in lowereukaryotes such as yeast or in prokaryotes such as bacteria. Potentiallysuitable yeast strains include Saccharomyces cerevisiae,Schizosaccharomyces pombe, Kluyveromyces strains, Candida, or any yeaststrain capable of expressing heterologous proteins. Potentially suitablebacterial strains include Escherichia coli, Bacillus subtilis,Salmonella typhimurium, or any bacterial strain capable of expressingheterologous proteins. If the protein is made in yeast or bacteria, itmay be necessary to modify the protein produced therein, for example byphosphorylation or glycosylation of the appropriate sites, in order toobtain the functional protein. Such covalent attachments may beaccomplished using known chemical or enzymatic methods.

The protein may also be produced by operably linking the isolatedpolynucleotide of the invention to suitable control sequences in one ormore insect expression vectors, and employing an insect expressionsystem. Materials and methods for baculovirus/insect cell expressionsystems are commercially available in kit form from, e.g., Invitrogen,San Diego, Calif., U.S.A. (the MaxBac® kit), and such methods are wellknown in the art, as described in Summers and Smith, Texas AgriculturalExperiment Station Bulletin No. 1555 (1987), incorporated herein byreference. As used herein, an insect cell capable of expressing apolynucleotide of the present invention is "transformed."

The protein of the invention may be prepared by culturing transformedhost cells under culture conditions suitable to express the recombinantprotein. The resulting expressed protein may then be purified from suchculture (i.e., from culture medium or cell extracts) using knownpurification processes, such as gel filtration and ion exchangechromatography. The purification of the protein may also include anaffinity column containing agents which will bind to the protein; one ormore column steps over such affinity resins as concanavalin A-agarose,heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; one or more stepsinvolving hydrophobic interaction chromatography using such resins asphenyl ether, butyl ether, or propyl ether; or immunoaffinitychromatography.

Alternatively, the protein of the invention may also be expressed in aform which will facilitate purification. For example, it may beexpressed as a fusion protein, such as those of maltose binding protein(MBP), glutathione-S-transferase (GST) or thioredoxin (TRX). Kits forexpression and purification of such fusion proteins are commerciallyavailable from New England BioLab (Beverly, Mass.), Pharmacia(Piscataway, N.J.) and In Vitrogen, respectively. The protein can alsobe tagged with an epitope and subsequently purified by using a specificantibody directed to such epitope. One such epitope ("Flag") iscommercially available from Kodak (New Haven, Conn.).

Finally, one or more reverse-phase high performance liquidchromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,e.g., silica gel having pendant methyl or other aliphatic groups, can beemployed to further purify the protein. Some or all of the foregoingpurification steps, in various combinations, can also be employed toprovide a substantially homogeneous isolated recombinant protein. Theprotein thus purified is substantially free of other mammalian proteinsand is defined in accordance with the present invention as an "isolatedprotein."

The protein of the invention may also be expressed as a product oftransgenic animals, e.g., as a component of the milk of transgenic cows,goats, pigs, or sheep which are characterized by somatic or germ cellscontaining a nucleotide sequence encoding the protein.

The protein may also be produced by known conventional chemicalsynthesis. Methods for constructing the proteins of the presentinvention by synthetic means are known to those skilled in the art. Thesynthetically-constructed protein sequences, by virtue of sharingprimary, secondary or tertiary structural and/or conformationalcharacteristics with proteins may possess biological properties incommon therewith, including protein activity. Thus, they may be employedas biologically active or immunological substitutes for natural,purified proteins in screening of therapeutic compounds and inimmunological processes for the development of antibodies.

The proteins provided herein also include proteins characterized byamino acid sequences similar to those of purified proteins but intowhich modification are naturally provided or deliberately engineered.For example, modifications in the peptide or DNA sequences can be madeby those skilled in the art using known techniques. Modifications ofinterest in the protein sequences may include the alteration,substitution, replacement, insertion or deletion of a selected aminoacid residue in the coding sequence. For example, one or more of thecysteine residues may be deleted or replaced with another amino acid toalter the conformation of the molecule. Techniques for such alteration,substitution, replacement, insertion or deletion are well known to thoseskilled in the art (see, e.g., U.S. Pat. No. 4,518,584). Preferably,such alteration, substitution, replacement, insertion or deletionretains the desired activity of the protein.

Other fragments and derivatives of the sequences of proteins which wouldbe expected to retain protein activity in whole or in part and may thusbe useful for screening or other immunological methodologies may also beeasily made by those skilled in the art given the disclosures herein.Such modifications are believed to be encompassed by the presentinvention.

USES AND BIOLOGICAL ACTIVITY

The polynucleotides and proteins of the present invention are expectedto exhibit one or more of the uses or biological activities (includingthose associated with assays cited herein) identified below. Uses oractivities described for proteins of the present invention may beprovided by administration or use of such proteins or by administrationor use of polynucleotides encoding such proteins (such as, for example,in gene therapies or vectors suitable for introduction of DNA).

Research Uses and Utilities

The polynucleotides provided by the present invention can be used by theresearch community for various purposes. The polynucleotides can be usedto express recombinant protein for analysis, characterization ortherapeutic use; as markers for tissues in which the correspondingprotein is preferentially expressed (either constitutively or at aparticular stage of tissue differentiation or development or in diseasestates); as molecular weight markers on Southern gels; as chromosomemarkers or tags (when labeled) to identify chromosomes or to map relatedgene positions; to compare with endogenous DNA sequences in patients toidentify potential genetic disorders; as probes to hybridize and thusdiscover novel, related DNA sequences; as a source of information toderive PCR primers for genetic fingerprinting; as a probe to"subtract-out" known sequences in the process of discovering other novelpolynucleotides; for selecting and making oligomers for attachment to a"gene chip" or other support, including for examination of expressionpatterns; to raise anti-protein antibodies using DNA immunizationtechniques; and as an antigen to raise anti-DNA antibodies or elicitanother immune response. Where the polynucleotide encodes a proteinwhich binds or potentially binds to another protein (such as, forexample, in a receptor-ligand interaction), the polynucleotide can alsobe used in interaction trap assays (such as, for example, that describedin Gyuris et al., Cell 75:791-803 (1993)) to identify polynucleotidesencoding the other protein with which binding occurs or to identifyinhibitors of the binding interaction.

The proteins provided by the present invention can similarly be used inassay to determine biological activity, including in a panel of multipleproteins for high-throughput screening; to raise antibodies or to elicitanother immune response; as a reagent (including the labeled reagent) inassays designed to quantitatively determine levels of the protein (orits receptor) in biological fluids; as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state); and, of course, to isolate correlative receptors orligands. Where the protein binds or potentially binds to another protein(such as, for example, in a receptor-ligand interaction), the proteincan be used to identify the other protein with which binding occurs orto identify inhibitors of the binding interaction. Proteins involved inthese binding interactions can also be used to screen for peptide orsmall molecule inhibitors or agonists of the binding interaction.

Any or all of these research utilities are capable of being developedinto reagent grade or kit format for commercialization as researchproducts.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include withoutlimitation "Molecular Cloning: A Laboratory Manual", 2d ed., Cold SpringHarbor Laboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatiseds., 1989, and "Methods in Enzymology: Guide to Molecular CloningTechniques", Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

Nutritional Uses

Polynucleotides and proteins of the present invention can also be usedas nutritional sources or supplements. Such uses include withoutlimitation use as a protein or amino acid supplement, use as a carbonsource, use as a nitrogen source and use as a source of carbohydrate. Insuch cases the protein or polynucleotide of the invention can be addedto the feed of a particular organism or can be administered as aseparate solid or liquid preparation, such as in the form of powder,pills, solutions, suspensions or capsules. In the case ofmicroorganisms, the protein or polynucleotide of the invention can beadded to the medium in or on which the microorganism is cultured.

Cytokine and Cell Proliferation/Differentiation Activity

A protein of the present invention may exhibit cytokine, cellproliferation (either inducing or inhibiting) or cell differentiation(either inducing or inhibiting) activity or may induce production ofother cytokines in certain cell populations. Many protein factorsdiscovered to date, including all known cytokines, have exhibitedactivity in one or more factor dependent cell proliferation assays, andhence the assays serve as a convenient confirmation of cytokineactivity. The activity of a protein of the present invention isevidenced by any one of a number of routine factor dependent cellproliferation assays for cell lines including, without limitation, 32D,DA2, DA1G, T10, B9, B9/11, BaF3, MC9/G, M+ (preB M+), 2E8, RB5, DA1,123, T1165, HT2, CTLL2, TF-1, Mo7e and CMK.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assays for T-cell or thymocyte proliferation include without limitationthose described in: Current Protocols in Immunology, Ed by J. E.Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W Strober,Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, InVitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7,Immunologic studies in Humans); Takai et al., J. Immunol. 137:3494-3500,1986; Bertagnolli et al., J. Immunol. 145:1706-1712, 1990; Bertagnolliet al., Cellular Immunology 133:327-341, 1991; Bertagnolli, et al., J.Immunol. 149:3778-3783, 1992; Bowman et al., J. Immunol. 152: 1756-1761,1994.

Assays for cytokine production and/or proliferation of spleen cells,lymph node cells or thymocytes include, without limitation, thosedescribed in: Polyclonal T cell stimulation, Kruisbeek, A. M. andShevach, E. M. In Current Protocols in Immunology. J.E.e.a. Coligan eds.Vol 1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto. 1994; andMeasurement of mouse and human Interferon γ, Schreiber, R. D. In CurrentProtocols in Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.8.1-6.8.8,John Wiley and Sons, Toronto. 1994.

Assays for proliferation and differentiation of hematopoietic andlymphopoietic cells include, without limitation, those described in:Measurement of Human and Murine Interleukin 2 and Interleukin 4,Bottomly, K., Davis, L. S. and Lipsky, P. E. In Current Protocols inImmunology. J.E.e.a. Coligan eds. Vol 1 pp. 6.3.1-6.3.12, John Wiley andSons, Toronto. 1991; deVries et al., J. Exp. Med. 173:1205-1211, 1991;Moreau et al., Nature 336:690-692, 1988; Greenberger et al., Proc. Natl.Acad. Sci. U.S.A. 80:2931-2938, 1983; Measurement of mouse and humaninterleukin 6--Nordan, R. In Current Protocols in Immunology. J.E.e.a.Coligan eds. Vol 1 pp. 6.6.1-6.6.5, John Wiley and Sons, Toronto. 1991;Smith et al., Proc. Natl. Acad. Sci. U.S.A. 83:1857-1861, 1986;Measurement of human Interleukin 11--Bennett, F., Giannotti, J., Clark,S. C. and Turner, K. J. In Current Protocols in Immunology. J.E.e.a.Coligan eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto. 1991;Measurement of mouse and human Interleukin 9--Ciarletta, A., Giannotti,J., Clark, S. C. and Turner, K. J. In Current Protocols in Immunology.J.E.e.a. Coligan eds. Vol 1 pp. 6.13.1, John Wiley and Sons, Toronto.1991.

Assays for T-cell clone responses to antigens (which will identify,among others, proteins that affect APC-T cell interactions as well asdirect T-cell effects by measuring proliferation and cytokineproduction) include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach, W Strober, Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 3, In Vitro assays for Mouse LymphocyteFunction; Chapter 6, Cytokines and their cellular receptors; Chapter 7,Immunologic studies in Humans); Weinberger et al., Proc. Natl. Acad.Sci. USA 77:6091-6095, 1980; Weinberger et al., Eur. J. Immun.11:405-411, 1981; Takai et al., J. Immunol. 137:3494-3500, 1986; Takaiet al., J. Immunol. 140:508-512, 1988.

Immune Stimulating or Suppressing Activity

A protein of the present invention may also exhibit immune stimulatingor immune suppressing activity, including without limitation theactivities for which assays are described herein. A protein may beuseful in the treatment of various immune deficiencies and disorders(including severe combined immunodeficiency (SCID)), e.g., in regulating(up or down) growth and proliferation of T and/or B lymphocytes, as wellas effecting the cytolytic activity of NK cells and other cellpopulations. These immune deficiencies may be genetic or be caused byviral (e.g., HIV) as well as bacterial or fungal infections, or mayresult from autoimmune disorders. More specifically, infectious diseasescauses by viral, bacterial, fungal or other infection may be treatableusing a protein of the present invention, including infections by HIV,hepatitis viruses, herpesviruses, mycobacteria, Leishmania spp., malariaspp. and various fungal infections such as candidiasis. Of course, inthis regard, a protein of the present invention may also be useful wherea boost to the immune system generally may be desirable, i.e., in thetreatment of cancer.

Autoimmune disorders which may be treated using a protein of the presentinvention include, for example, connective tissue disease, multiplesclerosis, systemic lupus erythematosus, rheumatoid arthritis,autoimmune pulmonary inflammation, Guillain-Barre syndrome, autoimmunethyroiditis, insulin dependent diabetes mellitis, myasthenia gravis,graft-versus-host disease and autoimmune inflammatory eye disease. Sucha protein of the present invention may also to be useful in thetreatment of allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems. Otherconditions, in which immune suppression is desired (including, forexample, organ transplantation), may also be treatable using a proteinof the present invention.

Using the proteins of the invention it may also be possible to immuneresponses, in a number of ways. Down regulation may be in the form ofinhibiting or blocking an immune response already in progress or mayinvolve preventing the induction of an immune response. The functions ofactivated T cells may be inhibited by suppressing T cell responses or byinducing specific tolerance in T cells, or both. Immunosuppression of Tcell responses is generally an active, non-antigen-specific, processwhich requires continuous exposure of the T cells to the suppressiveagent. Tolerance, which involves inducing non-responsiveness or anergyin T cells, is distinguishable from immunosuppression in that it isgenerally antigen-specific and persists after exposure to the tolerizingagent has ceased. Operationally, tolerance can be demonstrated by thelack of a T cell response upon reexposure to specific antigen in theabsence of the tolerizing agent.

Down regulating or preventing one or more antigen functions (includingwithout limitation B lymphocyte antigen functions (such as, for example,B7)), e.g., preventing high level lymphokine synthesis by activated Tcells, will be useful in situations of tissue, skin and organtransplantation and in graft-versus-host disease (GVHD). For example,blockage of T cell function should result in reduced tissue destructionin tissue transplantation. Typically, in tissue transplants, rejectionof the transplant is initiated through its recognition as foreign by Tcells, followed by an immune reaction that destroys the transplant. Theadministration of a molecule which inhibits or blocks interaction of aB7 lymphocyte antigen with its natural ligand(s) on immune cells (suchas a soluble, monomeric form of a peptide having B7-2 activity alone orin conjunction with a monomeric form of a peptide having an activity ofanother B lymphocyte antigen (e.g., B7-1, B7-3) or blocking antibody),prior to transplantation can lead to the binding of the molecule to thenatural ligand(s) on the immune cells without transmitting thecorresponding costimulatory signal. Blocking B lymphocyte antigenfunction in this matter prevents cytokine synthesis by immune cells,such as T cells, and thus acts as an immunosuppressant. Moreover, thelack of costimulation may also be sufficient to anergize the T cells,thereby inducing tolerance in a subject. Induction of long-termtolerance by B lymphocyte antigen-blocking reagents may avoid thenecessity of repeated administration of these blocking reagents. Toachieve sufficient immunosuppression or tolerance in a subject, it mayalso be necessary to block the function of a combination of B lymphocyteantigens.

The efficacy of particular blocking reagents in preventing organtransplant rejection or GVHD can be assessed using animal models thatare predictive of efficacy in humans. Examples of appropriate systemswhich can be used include allogeneic cardiac grafts in rats andxenogeneic pancreatic islet cell grafts in mice, both of which have beenused to examine the immunosuppressive effects of CTLA4Ig fusion proteinsin vivo as described in Lenschow et al., Science 257:789-792 (1992) andTurka et al., Proc. Natl. Acad. Sci USA, 89:11102-11105 (1992). Inaddition, murine models of GVHD (see Paul ed., Fundamental Immunology,Raven Press, New York, 1989, pp. 846-847) can be used to determine theeffect of blocking B lymphocyte antigen function in vivo on thedevelopment of that disease.

Blocking antigen function may also be therapeutically useful fortreating autoimmune diseases. Many autoimmune disorders are the resultof inappropriate activation of T cells that are reactive against selftissue and which promote the production of cytokines and autoantibodiesinvolved in the pathology of the diseases. Preventing the activation ofautoreactive T cells may reduce or eliminate disease symptoms.Administration of reagents which block costimulation of T cells bydisrupting receptor:ligand interactions of B lymphocyte antigens can beused to inhibit T cell activation and prevent production ofautoantibodies or T cell-derived cytokines which may be involved in thedisease process. Additionally, blocking reagents may induceantigen-specific tolerance of autoreactive T cells which could lead tolong-term relief from the disease. The efficacy of blocking reagents inpreventing or alleviating autoimmune disorders can be determined using anumber of well-characterized animal models of human autoimmune diseases.Examples include murine experimental autoimmune encephalitis, systemiclupus erythmatosis in MRL/lpr/lpr mice or NZB hybrid mice, murineautoimmune collagen arthritis, diabetes mellitus in NOD mice and BBrats, and murine experimental myasthenia gravis (see Paul ed.,Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).

Upregulation of an antigen function (preferably a B lymphocyte antigenfunction), as a means of up regulating immune responses, may also beuseful in therapy. Upregulation of immune responses may be in the formof enhancing an existing immune response or eliciting an initial immuneresponse. For example, enhancing an immune response through stimulatingB lymphocyte antigen function may be useful in cases of viral infection.In addition, systemic viral diseases such as influenza, the common cold,and encephalitis might be alleviated by the administration ofstimulatory forms of B lymphocyte antigens systemically.

Alternatively, anti-viral immune responses may be enhanced in aninfected patient by removing T cells from the patient, costimulating theT cells in vitro with viral antigen-pulsed APCs either expressing apeptide of the present invention or together with a stimulatory form ofa soluble peptide of the present invention and reintroducing the invitro activated T cells into the patient. Another method of enhancinganti-viral immune responses would be to isolate infected cells from apatient, transfect them with a nucleic acid encoding a protein of thepresent invention as described herein such that the cells express all ora portion of the protein on their surface, and reintroduce thetransfected cells into the patient. The infected cells would now becapable of delivering a costimulatory signal to, and thereby activate, Tcells in vivo.

In another application, up regulation or enhancement of antigen function(preferably B lymphocyte antigen function) may be useful in theinduction of tumor immunity. Tumor cells (e.g., sarcoma, melanoma,lymphoma, leukemia, neuroblastoma, carcinoma) transfected with a nucleicacid encoding at least one peptide of the present invention can beadministered to a subject to overcome tumor-specific tolerance in thesubject. If desired, the tumor cell can be transfected to express acombination of peptides. For example, tumor cells obtained from apatient can be transfected ex vivo with an expression vector directingthe expression of a peptide having B7-2-like activity alone, or inconjunction with a peptide having B7-1-like activity and/or B7-3-likeactivity. The transfected tumor cells are returned to the patient toresult in expression of the peptides on the surface of the transfectedcell. Alternatively, gene therapy techniques can be used to target atumor cell for transfection in vivo.

The presence of the peptide of the present invention having the activityof a B lymphocyte antigen(s) on the surface of the tumor cell providesthe necessary costimulation signal to T cells to induce a T cellmediated immune response against the transfected tumor cells. Inaddition, tumor cells which lack MHC class I or MHC class II molecules,or which fail to reexpress sufficient amounts of MHC class I or MHCclass II molecules, can be transfected with nucleic acid encoding all ora portion of (e.g., a cytoplasmic-domain truncated portion) of an MHCclass I α chain protein and β₂ microglobulin protein or an MHC class IIα chain protein and an MHC class II β chain protein to thereby expressMHC class I or MHC class II proteins on the cell surface. Expression ofthe appropriate class I or class II MHC in conjunction with a peptidehaving the activity of a B lymphocyte antigen (e.g., B7-1, B7-2, B7-3)induces a T cell mediated immune response against the transfected tumorcell. Optionally, a gene encoding an antisense construct which blocksexpression of an MHC class II associated protein, such as the invariantchain, can also be cotransfected with a DNA encoding a peptide havingthe activity of a B lymphocyte antigen to promote presentation of tumorassociated antigens and induce tumor specific immunity. Thus, theinduction of a T cell mediated immune response in a human subject may besufficient to overcome tumor-specific tolerance in the subject.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Suitable assays for thymocyte or splenocyte cytotoxicity include,without limitation, those described in: Current Protocols in Immunology,Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, WStrober, Pub. Greene Publishing Associates and Wiley-Interscience(Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19;Chapter 7, Immunologic studies in Humans); Herrmann et al., Proc. Natl.Acad. Sci. USA 78:2488-2492, 1981; Herrmann et al., J. Immunol.128:1968-1974, 1982; Handa et al., J. Immunol. 135:1564-1572, 1985;Takai et al., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.140:508-512, 1988; Herrmann et al., Proc. Natl. Acad. Sci. USA78:2488-2492, 1981; Herrmann et al., J. Immunol. 128:1968-1974, 1982;Handa et al., J. Immunol. 135:1564-1572, 1985; Takai et al., J. Immunol.137:3494-3500, 1986; Bowmanet al., J. Virology 61:1992-1998; Takai etal., J. Immunol. 140:508-512, 1988; Bertagnolli et al., CellularImmunology 133:327-341, 1991; Brown et al., J. Immunol. 153:3079-3092,1994.

Assays for T-cell-dependent immunoglobulin responses and isotypeswitching (which will identify, among others, proteins that modulateT-cell dependent antibody responses and that affect Th1/Th2 profiles)include, without limitation, those described in: Maliszewski, J.Immunol. 144:3028-3033, 1990; and Assays for B cell function: In vitroantibody production, Mond, J. J. and Brunswick, M. In Current Protocolsin Immunology. J.E.e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wileyand Sons, Toronto. 1994.

Mixed lymphocyte reaction (MLR) assays (which will identify, amongothers, proteins that generate predominantly Th1 and CTL responses)include, without limitation, those described in: Current Protocols inImmunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M.Shevach, W Strober, Pub. Greene Publishing Associates andWiley-Interscience (Chapter 3, In Vitro assays for Mouse LymphocyteFunction 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai etal., J. Immunol. 137:3494-3500, 1986; Takai et al., J. Immunol.140:508-512, 1988; Bertagnolli et al., J. Immunol. 149:3778-3783, 1992.

Dendritic cell-dependent assays (which will identify, among others,proteins expressed by dendritic cells that activate naive T-cells)include, without limitation, those described in: Guery et al., J.Immunol. 134:536-544, 1995; Inaba et al., Journal of ExperimentalMedicine 173:549-559, 1991; Macatonia et al., Journal of Immunology154:5071-5079, 1995; Porgador et al., Journal of Experimental Medicine182:255-260, 1995; Nair et al., Journal of Virology 67:4062-4069, 1993;Huang et al., Science 264:961-965, 1994; Macatonia et al., Journal ofExperimental Medicine 169:1255-1264, 1989; Bhardwaj et al., Journal ofClinical Investigation 94:797-807, 1994; and Inaba et al., Journal ofExperimental Medicine 172:631-640, 1990.

Assays for lymphocyte survival/apoptosis (which will identify, amongothers, proteins that prevent apoptosis after superantigen induction andproteins that regulate lymphocyte homeostasis) include, withoutlimitation, those described in: Darzynkiewicz et al., Cytometry13:795-808, 1992; Gorczyca et al., Leukemia 7:659-670, 1993; Gorczyca etal., Cancer Research 53:1945-1951, 1993; Itoh et al., Cell 66:233-243,1991; Zacharchuk, Journal of Immunology 145:4037-4045, 1990; Zamai etal., Cytometry 14:891-897, 1993; Gorczyca et al., International Journalof Oncology 1:639-648, 1992.

Assays for proteins that influence early steps of T-cell commitment anddevelopment include, without limitation, those described in: Antica etal., Blood 84:111-117, 1994; Fine et al., Cellular Immunology155:111-122, 1994; Galy et al., Blood 85:2770-2778, 1995; Toki et al.,Proc. Nat. Acad Sci. USA 88:7548-7551, 1991.

Hematopoiesis Regulating Activity

A protein of the present invention may be useful in regulation ofhematopoiesis and, consequently, in the treatment of myeloid or lymphoidcell deficiencies. Even marginal biological activity in support ofcolony forming cells or of factor-dependent cell lines indicatesinvolvement in regulating hematopoiesis, e.g. in supporting the growthand proliferation of erythroid progenitor cells alone or in combinationwith other cytokines, thereby indicating utility, for example, intreating various anemias or for use in conjunction withirradiation/chemotherapy to stimulate the production of erythroidprecursors and/or erythroid cells; in supporting the growth andproliferation of myeloid cells such as granulocytes andmonocytes/macrophages (i.e., traditional CSF activity) useful, forexample, in conjunction with chemotherapy to prevent or treat consequentmyelo-suppression; in supporting the growth and proliferation ofmegakaryocytes and consequently of platelets thereby allowing preventionor treatment of various platelet disorders such as thrombocytopenia, andgenerally for use in place of or complimentary to platelet transfusions;and/or in supporting the growth and proliferation of hematopoietic stemcells which are capable of maturing to any and all of theabove-mentioned hematopoietic cells and therefore find therapeuticutility in various stem cell disorders (such as those usually treatedwith transplantation, including, without limitation, aplastic anemia andparoxysmal nocturnal hemoglobinuria), as well as in repopulating thestem cell compartment post irradiation/chemotherapy, either in-vivo orex-vivo (i.e., in conjunction with bone marrow transplantation or withperipheral progenitor cell transplantation (homologous or heterologous))as normal cells or genetically manipulated for gene therapy.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Suitable assays for proliferation and differentiation of varioushematopoietic lines are cited above.

Assays for embryonic stem cell differentiation (which will identify,among others, proteins that influence embryonic differentiationhematopoiesis) include, without limitation, those described in:Johansson et al. Cellular Biology 15:141-151, 1995; Keller et al.,Molecular and Cellular Biology 13:473-486, 1993; McClanahan et al.,Blood 81:2903-2915, 1993.

Assays for stem cell survival and differentiation (which will identify,among others, proteins that regulate lympho-hematopoiesis) include,without limitation, those described in: Methylcellulose colony formingassays, Freshney, M. G. In Culture of Hematopoietic Cells. R. I.Freshney, et al. eds. Vol pp. 265-268, Wiley-Liss, Inc., New York, N.Y.1994; Hirayama et al., Proc. Natl. Acad. Sci. USA 89:5907-5911, 1992;Primitive hematopoietic colony forming cells with high proliferativepotential, McNiece, I. K. and Briddell, R. A. In Culture ofHematopoietic Cells. R. I. Freshney, et al. eds. Vol pp. 23-39,Wiley-Liss, Inc., New York, N.Y. 1994; Neben et al., ExperimentalHematology 22:353-359, 1994; Cobblestone area forming cell assay,Ploemacher, R. E. In Culture of Hematopoietic Cells. R. I. Freshney, etal. eds. Vol pp. 1-21, Wiley-Liss, Inc., New York, N.Y. 1994; Long termbone marrow cultures in the presence of stromal cells, Spooncer, E.,Dexter, M. and Allen, T. In Culture of Hematopoietic Cells. R. I.Freshney, et al. eds. Vol pp. 163-179, Wiley-Liss, Inc., New York, N.Y.1994; Long term culture initiating cell assay, Sutherland, H. J. InCulture of Hematopoietic Cells. R. I. Freshney, et al. eds. Vol pp.139-162, Wiley-Liss, Inc., New York, N.Y. 1994.

Tissue Growth Activity

A protein of the present invention also may have utility in compositionsused for bone, cartilage, tendon, ligament and/or nerve tissue growth orregeneration, as well as for wound healing and tissue repair andreplacement, and in the treatment of burns, incisions and ulcers.

A protein of the present invention, which induces cartilage and/or bonegrowth in circumstances where bone is not normally formed, hasapplication in the healing of bone fractures and cartilage damage ordefects in humans and other animals. Such a preparation employing aprotein of the invention may have prophylactic use in closed as well asopen fracture reduction and also in the improved fixation of artificialjoints. De novo bone formation induced by an osteogenic agentcontributes to the repair of congenital, trauma induced, or oncologicresection induced craniofacial defects, and also is useful in cosmeticplastic surgery.

A protein of this invention may also be used in the treatment ofperiodontal disease, and in other tooth repair processes. Such agentsmay provide an environment to attract bone-forming cells, stimulategrowth of bone-forming cells or induce differentiation of progenitors ofbone-forming cells. A protein of the invention may also be useful in thetreatment of osteoporosis or osteoarthritis, such as through stimulationof bone and/or cartilage repair or by blocking inflammation or processesof tissue destruction (collagenase activity, osteoclast activity, etc.)mediated by inflammatory processes.

Another category of tissue regeneration activity that may beattributable to the protein of the present invention is tendon/ligamentformation. A protein of the present invention, which inducestendon/ligament-like tissue or other tissue formation in circumstanceswhere such tissue is not normally formed, has application in the healingof tendon or ligament tears, deformities and other tendon or ligamentdefects in humans and other animals. Such a preparation employing atendon/ligament-like tissue inducing protein may have prophylactic usein preventing damage to tendon or ligament tissue, as well as use in theimproved fixation of tendon or ligament to bone or other tissues, and inrepairing defects to tendon or ligament tissue. De novotendon/ligament-like tissue formation induced by a composition of thepresent invention contributes to the repair of congenital, traumainduced, or other tendon or ligament defects of other origin, and isalso useful in cosmetic plastic surgery for attachment or repair oftendons or ligaments. The compositions of the present invention mayprovide an environment to attract tendon- or ligament-forming cells,stimulate growth of tendon- or ligament-forming cells, inducedifferentiation of progenitors of tendon- or ligament-forming cells, orinduce growth of tendon/ligament cells or progenitors ex vivo for returnin vivo to effect tissue repair. The compositions of the invention mayalso be useful in the treatment of tendinitis, carpal tunnel syndromeand other tendon or ligament defects. The compositions may also includean appropriate matrix and/or sequestering agent as a carrier as is wellknown in the art.

The protein of the present invention may also be useful forproliferation of neural cells and for regeneration of nerve and braintissue, i.e. for the treatment of central and peripheral nervous systemdiseases and neuropathies, as well as mechanical and traumaticdisorders, which involve degeneration, death or trauma to neural cellsor nerve tissue. More specifically, a protein may be used in thetreatment of diseases of the peripheral nervous system, such asperipheral nerve injuries, peripheral neuropathy and localizedneuropathies, and central nervous system diseases, such as Alzheimer's,Parkinson's disease, Huntington's disease, amyotrophic lateralsclerosis, and Shy-Drager syndrome. Further conditions which may betreated in accordance with the present invention include mechanical andtraumatic disorders, such as spinal cord disorders, head trauma andcerebrovascular diseases such as stroke. Peripheral neuropathiesresulting from chemotherapy or other medical therapies may also betreatable using a protein of the invention.

Proteins of the invention may also be useful to promote better or fasterclosure of non-healing wounds, including without limitation pressureulcers, ulcers associated with vascular insufficiency, surgical andtraumatic wounds, and the like.

It is expected that a protein of the present invention may also exhibitactivity for generation or regeneration of other tissues, such as organs(including, for example, pancreas, liver, intestine, kidney, skin,endothelium), muscle (smooth, skeletal or cardiac) and vascular(including vascular endothelium) tissue, or for promoting the growth ofcells comprising such tissues. Part of the desired effects may be byinhibition or modulation of fibrotic scarring to allow normal tissue toregenerate. A protein of the invention may also exhibit angiogenicactivity.

A protein of the present invention may also be useful for gut protectionor regeneration and treatment of lung or liver fibrosis, reperfusioninjury in various tissues, and conditions resulting from systemiccytokine damage.

A protein of the present invention may also be useful for promoting orinhibiting differentiation of tissues described above from precursortissues or cells; or for inhibiting the growth of tissues describedabove.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assays for tissue generation activity include, without limitation, thosedescribed in: International Patent Publication No. WO95/16035 (bone,cartilage, tendon); International Patent Publication No. WO95/05846(nerve, neuronal); International Patent Publication No. WO91/07491(skin, endothelium).

Assays for wound healing activity include, without limitation, thosedescribed in: Winter, Epidermal Wound Healing, pps. 71-112 (Maibach, H Iand Rovee, D T, eds.), Year Book Medical Publishers, Inc., Chicago, asmodified by Eaglstein and Mertz, J. Invest. Dermatol 71:382-84 (1978).

Activin/Inhibin Activity

A protein of the present invention may also exhibit activin- orinhibin-related activities. Inhibins are characterized by their abilityto inhibit the release of follicle stimulating hormone (FSH), whileactivins and are characterized by their ability to stimulate the releaseof follicle stimulating hormone (FSH). Thus, a protein of the presentinvention, alone or in heterodimers with a member of the inhibin αfamily, may be useful as a contraceptive based on the ability ofinhibins to decrease fertility in female mammals and decreasespermatogenesis in male mammals. Administration of sufficient amounts ofother inhibins can induce infertility in these mammals. Alternatively,the protein of the invention, as a homodimer or as a heterodimer withother protein subunits of the inhibin-β group, may be useful as afertility inducing therapeutic, based upon the ability of activinmolecules in stimulating FSH release from cells of the anteriorpituitary. See, for example, U.S. Pat. No. 4,798,885. A protein of theinvention may also be useful for advancement of the onset of fertilityin sexually immature mammals, so as to increase the lifetimereproductive performance of domestic animals such as cows, sheep andpigs.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assays for activin/inhibin activity include, without limitation, thosedescribed in: Vale et al., Endocrinology 91:562-572, 1972; Ling et al.,Nature 321:779-782, 1986; Vale et al., Nature 321:776-779, 1986; Masonet al., Nature 318:659-663, 1985; Forage et al., Proc. Natl. Acad. Sci.USA 83:3091-3095, 1986.

Chemotactic/Chemokinetic Activity

A protein of the present invention may have chemotactic or chemokineticactivity (e.g., act as a chemokine) for mammalian cells, including, forexample, monocytes, fibroblasts, neutrophils, T-cells, mast cells,eosinophils, epithelial and/or endothelial cells. Chemotactic andchemokinetic proteins can be used to mobilize or attract a desired cellpopulation to a desired site of action. Chemotactic or chemokineticproteins provide particular advantages in treatment of wounds and othertrauma to tissues, as well as in treatment of localized infections. Forexample, attraction of lymphocytes, monocytes or neutrophils to tumorsor sites of infection may result in improved immune responses againstthe tumor or infecting agent.

A protein or peptide has chemotactic activity for a particular cellpopulation if it can stimulate, directly or indirectly, the directedorientation or movement of such cell population. Preferably, the proteinor peptide has the ability to directly stimulate directed movement ofcells. Whether a particular protein has chemotactic activity for apopulation of cells can be readily determined by employing such proteinor peptide in any known assay for cell chemotaxis.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assays for chemotactic activity (which will identify proteins thatinduce or prevent chemotaxis) consist of assays that measure the abilityof a protein to induce the migration of cells across a membrane as wellas the ability of a protein to induce the adhesion of one cellpopulation to another cell population. Suitable assays for movement andadhesion include, without limitation, those described in: CurrentProtocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H.Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associatesand Wiley-Interscience (Chapter 6.12, Measurement of alpha and betaChemokines 6.12.1-6.12.28; Taub et al. J. Clin. Invest. 95:1370-1376,1995; Lind et al. APMIS 103:140-146, 1995; Muller et al Eur. J. Immunol.25: 1744-1748; Gruber et al. J. of Immunol. 152:5860-5867, 1994;Johnston et al. J. of Immunol. 153: 1762-1768, 1994.

Hemostatic and Thrombolytic Activity

A protein of the invention may also exhibit hemostatic or thrombolyticactivity. As a result, such a protein is expected to be useful intreatment of various coagulation disorders (including hereditarydisorders, such as hemophilias) or to enhance coagulation and otherhemostatic events in treating wounds resulting from trauma, surgery orother causes. A protein of the invention may also be useful fordissolving or inhibiting formation of thromboses and for treatment andprevention of conditions resulting therefrom (such as, for example,infarction of cardiac and central nervous system vessels (e.g., stroke).

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Assay for hemostatic and thrombolytic activity include, withoutlimitation, those described in: Linet et al., J. Clin. Pharmacol.26:131-140, 1986; Burdick et al., Thrombosis Res. 45:413-419, 1987;Humphrey et al., Fibrinolysis 5:71-79 (1991); Schaub, Prostaglandins35:467-474, 1988.

Receptor/Ligand Activity

A protein of the present invention may also demonstrate activity asreceptors, receptor ligands or inhibitors or agonists of receptor/ligandinteractions. Examples of such receptors and ligands include, withoutlimitation, cytokine receptors and their ligands, receptor kinases andtheir ligands, receptor phosphatases and their ligands, receptorsinvolved in cell-cell interactions and their ligands (including withoutlimitation, cellular adhesion molecules (such as selectins, integrinsand their ligands) and receptor/ligand pairs involved in antigenpresentation, antigen recognition and development of cellular andhumoral immune responses). Receptors and ligands are also useful forscreening of potential peptide or small molecule inhibitors of therelevant receptor/ligand interaction. A protein of the present invention(including, without limitation, fragments of receptors and ligands) maythemselves be useful as inhibitors of receptor/ligand interactions.

The activity of a protein of the invention may, among other means, bemeasured by the following methods:

Suitable assays for receptor-ligand activity include without limitationthose described in: Current Protocols in Immunology, Ed by J. E.Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober,Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 7.28,Measurement of Cellular Adhesion under static conditions7.28.1-7.28.22), Takai et al., Proc. Natl. Acad. Sci. USA 84:6864-6868,1987; Bierer et al., J. Exp. Med. 168:1145-1156, 1988; Rosenstein etal., J. Exp. Med. 169:149-160 1989; Stoltenborg et al., J. Immunol.Methods 175:59-68, 1994; Stitt et al., Cell 80:661-670, 1995.

Anti-Inflammatory Activity

Proteins of the present invention may also exhibit anti-inflammatoryactivity. The anti-inflammatory activity may be achieved by providing astimulus to cells involved in the inflammatory response, by inhibitingor promoting cell-cell interactions (such as, for example, celladhesion), by inhibiting or promoting chemotaxis of cells involved inthe inflammatory process, inhibiting or promoting cell extravasation, orby stimulating or suppressing production of other factors which moredirectly inhibit or promote an inflammatory response. Proteinsexhibiting such activities can be used to treat inflammatory conditionsincluding chronic or acute conditions), including without limitationinflammation associated with infection (such as septic shock, sepsis orsystemic inflammatory response syndrome (SIRS)), ischemia-reperfusioninjury, endotoxin lethality, arthritis, complement-mediated hyperacuterejection, nephritis, cytokine or chemokine-induced lung injury,inflammatory bowel disease, Crohn's disease or resulting from overproduction of cytokines such as TNF or IL-1. Proteins of the inventionmay also be useful to treat anaphylaxis and hypersensitivity to anantigenic substance or material.

Tumor Inhibition Activity

In addition to the activities described above for immunologicaltreatment or prevention of tumors, a protein of the invention mayexhibit other anti-tumor activities. A protein may inhibit tumor growthdirectly or indirectly (such as, for example, via ADCC). A protein mayexhibit its tumor inhibitory activity by acting on tumor tissue or tumorprecursor tissue, by inhibiting formation of tissues necessary tosupport tumor growth (such as, for example, by inhibiting angiogenesis),by causing production of other factors, agents or cell types whichinhibit tumor growth, or by suppressing, eliminating or inhibitingfactors, agents or cell types which promote tumor growth.

Other Activities

A protein of the invention may also exhibit one or more of the followingadditional activities or effects: inhibiting the growth, infection orfunction of, or killing, infectious agents, including, withoutlimitation, bacteria, viruses, fungi and other parasites; effecting(suppressing or enhancing) bodily characteristics, including, withoutlimitation, height, weight, hair color, eye color, skin, fat to leanratio or other tissue pigmentation, or organ or body part size or shape(such as, for example, breast augmentation or diminution, change in boneform or shape); effecting biorhythms or caricadic cycles or rhythms;effecting the fertility of male or female subjects; effecting themetabolism, catabolism, anabolism, processing, utilization, storage orelimination of dietary fat, lipid, protein, carbohydrate, vitamins,minerals, cofactors or other nutritional factors or component(s);effecting behavioral characteristics, including, without limitation,appetite, libido, stress, cognition (including cognitive disorders),depression (including depressive disorders) and violent behaviors;providing analgesic effects or other pain reducing effects; promotingdifferentiation and growth of embryonic stem cells in lineages otherthan hematopoietic lineages; hormonal or endocrine activity; in the caseof enzymes, correcting deficiencies of the enzyme and treatingdeficiency-related diseases; treatment of hyperproliferative disorders(such as, for example, psoriasis); immunoglobulin-like activity (suchas, for example, the ability to bind antigens or complement); and theability to act as an antigen in a vaccine composition to raise an immuneresponse against such protein or another material or entity which iscross-reactive with such protein.

ADMINISTRATION AND DOSING

A protein of the present invention (from whatever source derived,including without limitation from recombinant and non-recombinantsources) may be used in a pharmaceutical composition when combined witha pharmaceutically acceptable carrier. Such a composition may alsocontain (in addition to protein and a carrier) diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials well known inthe art. The term "pharmaceutically acceptable" means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the active ingredient(s). The characteristics ofthe carrier will depend on the route of administration. Thepharmaceutical composition of the invention may also contain cytokines,lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF,IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,IL-12, IL-13, IL-14, IL-15, IFN, TNF0, TNF1, TNF2, G-CSF, Meg-CSF,thrombopoietin, stem cell factor, and erythropoietin. The pharmaceuticalcomposition may further contain other agents which either enhance theactivity of the protein or compliment its activity or use in treatment.Such additional factors and/or agents may be included in thepharmaceutical composition to produce a synergistic effect with proteinof the invention, or to minimize side effects. Conversely, protein ofthe present invention may be included in formulations of the particularcytokine, lymphokine, other hematopoietic factor, thrombolytic oranti-thrombotic factor, or anti-inflammatory agent to minimize sideeffects of the cytokine, lymphokine, other hematopoietic factor,thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.

A protein of the present invention may be active in multimers (e.g.,heterodimers or homodimers) or complexes with itself or other proteins.As a result, pharmaceutical compositions of the invention may comprise aprotein of the invention in such multimeric or complexed form.

The pharmaceutical composition of the invention may be in the form of acomplex of the protein(s) of present invention along with protein orpeptide antigens. The protein and/or peptide antigen will deliver astimulatory signal to both B and T lymphocytes. B lymphocytes willrespond to antigen through their surface immunoglobulin receptor. Tlymphocytes will respond to antigen through the T cell receptor (TCR)following presentation of the antigen by MHC proteins. MHC andstructurally related proteins including those encoded by class I andclass II MHC genes on host cells will serve to present the peptideantigen(s) to T lymphocytes. The antigen components could also besupplied as purified MHC-peptide complexes alone or with co-stimulatorymolecules that can directly signal T cells. Alternatively antibodiesable to bind surface immunolgobulin and other molecules on B cells aswell as antibodies able to bind the TCR and other molecules on T cellscan be combined with the pharmaceutical composition of the invention.

The pharmaceutical composition of the invention may be in the form of aliposome in which protein of the present invention is combined, inaddition to other pharmaceutically acceptable carriers, with amphipathicagents such as lipids which exist in aggregated form as micelles,insoluble monolayers, liquid crystals, or lamellar layers in aqueoussolution. Suitable lipids for liposomal formulation include, withoutlimitation, monoglycerides, diglycerides, sulfatides, lysolecithin,phospholipids, saponin, bile acids, and the like. Preparation of suchliposomal formulations is within the level of skill in the art, asdisclosed, for example, in U.S. Pat. No. 4,235,871; U.S. Pat. No.4,501,728; U.S. Pat. No. 4,837,028; and U.S. Pat. No. 4,737,323, all ofwhich are incorporated herein by reference.

As used herein, the term "therapeutically effective amount" means thetotal amount of each active component of the pharmaceutical compositionor method that is sufficient to show a meaningful patient benefit, i.e.,treatment, healing, prevention or amelioration of the relevant medicalcondition, or an increase in rate of treatment, healing, prevention oramelioration of such conditions. When applied to an individual activeingredient, administered alone, the term refers to that ingredientalone. When applied to a combination, the term refers to combinedamounts of the active ingredients that result in the therapeutic effect,whether administered in combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, atherapeutically effective amount of protein of the present invention isadministered to a mammal having a condition to be treated. Protein ofthe present invention may be administered in accordance with the methodof the invention either alone or in combination with other therapiessuch as treatments employing cytokines, lymphokines or otherhematopoietic factors. When co-administered with one or more cytokines,lymphokines or other hematopoietic factors, protein of the presentinvention may be administered either simultaneously with thecytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolyticor anti-thrombotic factors, or sequentially. If administeredsequentially, the attending physician will decide on the appropriatesequence of administering protein of the present invention incombination with cytokine(s), lymphokine(s), other hematopoieticfactor(s), thrombolytic or anti-thrombotic factors.

Administration of protein of the present invention used in thepharmaceutical composition or to practice the method of the presentinvention can be carried out in a variety of conventional ways, such asoral ingestion, inhalation, topical application or cutaneous,subcutaneous, intraperitoneal, parenteral or intravenous injection.Intravenous administration to the patient is preferred.

When a therapeutically effective amount of protein of the presentinvention is administered orally, protein of the present invention willbe in the form of a tablet, capsule, powder, solution or elixir. Whenadministered in tablet form, the pharmaceutical composition of theinvention may additionally contain a solid carrier such as a gelatin oran adjuvant. The tablet, capsule, and powder contain from about 5 to 95%protein of the present invention, and preferably from about 25 to 90%protein of the present invention. When administered in liquid form, aliquid carrier such as water, petroleum, oils of animal or plant originsuch as peanut oil, mineral oil, soybean oil, or sesame oil, orsynthetic oils may be added. The liquid form of the pharmaceuticalcomposition may further contain physiological saline solution, dextroseor other saccharide solution, or glycols such as ethylene glycol,propylene glycol or polyethylene glycol. When administered in liquidform, the pharmaceutical composition contains from about 0.5 to 90% byweight of protein of the present invention, and preferably from about 1to 50% protein of the present invention.

When a therapeutically effective amount of protein of the presentinvention is administered by intravenous, cutaneous or subcutaneousinjection, protein of the present invention will be in the form of apyrogen-free, parenterally acceptable aqueous solution. The preparationof such parenterally acceptable protein solutions, having due regard topH, isotonicity, stability, and the like, is within the skill in theart. A preferred pharmaceutical composition for intravenous, cutaneous,or subcutaneous injection should contain, in addition to protein of thepresent invention, an isotonic vehicle such as Sodium ChlorideInjection, Ringer's Injection, Dextrose Injection, Dextrose and SodiumChloride Injection, Lactated Ringer's Injection, or other vehicle asknown in the art. The pharmaceutical composition of the presentinvention may also contain stabilizers, preservatives, buffers,antioxidants, or other additives known to those of skill in the art.

The amount of protein of the present invention in the pharmaceuticalcomposition of the present invention will depend upon the nature andseverity of the condition being treated, and on the nature of priortreatments which the patient has undergone. Ultimately, the attendingphysician will decide the amount of protein of the present inventionwith which to treat each individual patient. Initially, the attendingphysician will administer low doses of protein of the present inventionand observe the patient's response. Larger doses of protein of thepresent invention may be administered until the optimal therapeuticeffect is obtained for the patient, and at that point the dosage is notincreased further. It is contemplated that the various pharmaceuticalcompositions used to practice the method of the present invention shouldcontain about 0.01 μg to about 100 mg (preferably about 0.1 μg to about10 mg, more preferably about 0.01 μg to about 1 mg) of protein of thepresent invention per kg body weight.

The duration of intravenous therapy using the pharmaceutical compositionof the present invention will vary, depending on the severity of thedisease being treated and the condition and potential idiosyncraticresponse of each individual patient. It is contemplated that theduration of each application of the protein of the present inventionwill be in the range of 12 to 24 hours of continuous intravenousadministration. Ultimately the attending physician will decide on theappropriate duration of intravenous therapy using the pharmaceuticalcomposition of the present invention.

Protein of the invention may also be used to immunize animals to obtainpolyclonal and monoclonal antibodies which specifically react with theprotein. Such antibodies may be obtained using either the entire proteinor fragments thereof as an immunogen. The peptide immunogensadditionally may contain a cysteine residue at the carboxyl terminus,and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH).Methods for synthesizing such peptides are known in the art, forexample, as in R. P. Merrifield, J. Amer. Chem. Soc. 85, 2149-2154(1963); J. L. Krstenansky, et al., FEBS Lett. 211, 10 (1987). Monoclonalantibodies binding to the protein of the invention may be usefuldiagnostic agents for the immunodetection of the protein. Neutralizingmonoclonal antibodies binding to the protein may also be usefultherapeutics for both conditions associated with the protein and also inthe treatment of some forms of cancer where abnormal expression of theprotein is involved. In the case of cancerous cells or leukemic cells,neutralizing monoclonal antibodies against the protein may be useful indetecting and preventing the metastatic spread of the cancerous cells,which may be mediated by the protein.

For compositions of the present invention which are useful for bone,cartilage, tendon or ligament regeneration, the therapeutic methodincludes administering the composition topically, systematically, orlocally as an implant or device. When administered, the therapeuticcomposition for use in this invention is, of course, in a pyrogen-free,physiologically acceptable form. Further, the composition may desirablybe encapsulated or injected in a viscous form for delivery to the siteof bone, cartilage or tissue damage. Topical administration may besuitable for wound healing and tissue repair. Therapeutically usefulagents other than a protein of the invention which may also optionallybe included in the composition as described above, may alternatively oradditionally, be administered simultaneously or sequentially with thecomposition in the methods of the invention. Preferably for bone and/orcartilage formation, the composition would include a matrix capable ofdelivering the protein-containing composition to the site of bone and/orcartilage damage, providing a structure for the developing bone andcartilage and optimally capable of being resorbed into the body. Suchmatrices may be formed of materials presently in use for other implantedmedical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the compositionswill define the appropriate formulation. Potential matrices for thecompositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid,polyglycolic acid and polyanhydrides. Other potential materials arebiodegradable and biologically well-defined, such as bone or dermalcollagen. Further matrices are comprised of pure proteins orextracellular matrix components. Other potential matrices arenonbiodegradable and chemically defined, such as sintered hydroxapatite,bioglass, aluminates, or other ceramics. Matrices may be comprised ofcombinations of any of the above mentioned types of material, such aspolylactic acid and hydroxyapatite or collagen and tricalciumphosphate.The bioceramics may be altered in composition, such as incalcium-aluminate-phosphate and processing to alter pore size, particlesize, particle shape, and biodegradability.

Presently preferred is a 50:50 (mole weight) copolymer of lactic acidand glycolic acid in the form of porous particles having diametersranging from 150 to 800 microns. In some applications, it will be usefulto utilize a sequestering agent, such as carboxymethyl cellulose orautologous blood clot, to prevent the protein compositions fromdisassociating from the matrix.

A preferred family of sequestering agents is cellulosic materials suchas alkylcelluloses (including hydroxyalkylcelluloses), includingmethylcellulose, ethylcellulose, hydroxyethylcellulose,hydroxypropylcellulose, hydroxypropyl-methylcellulose, andcarboxymethylcellulose, the most preferred being cationic salts ofcarboxymethylcellulose (CMC). Other preferred sequestering agentsinclude hyaluronic acid, sodium alginate, poly(ethylene glycol),polyoxyethylene oxide, carboxyvinyl polymer and poly(vinyl alcohol). Theamount of sequestering agent useful herein is 0.5-20 wt %, preferably1-10 wt % based on total formulation weight, which represents the amountnecessary to prevent desorbtion of the protein from the polymer matrixand to provide appropriate handling of the composition, yet not so muchthat the progenitor cells are prevented from infiltrating the matrix,thereby providing the protein the opportunity to assist the osteogenicactivity of the progenitor cells.

In further compositions, proteins of the invention may be combined withother agents beneficial to the treatment of the bone and/or cartilagedefect, wound, or tissue in question. These agents include variousgrowth factors such as epidermal growth factor (EGF), platelet derivedgrowth factor (PDGF), transforming growth factors (TGF-α and TGF-β), andinsulin-like growth factor (IGF).

The therapeutic compositions are also presently valuable for veterinaryapplications. Particularly domestic animals and thoroughbred horses, inaddition to humans, are desired patients for such treatment withproteins of the present invention.

The dosage regimen of a protein-containing pharmaceutical composition tobe used in tissue regeneration will be determined by the attendingphysician considering various factors which modify the action of theproteins, e.g., amount of tissue weight desired to be formed, the siteof damage, the condition of the damaged tissue, the size of a wound,type of damaged tissue (e.g., bone), the patient's age, sex, and diet,the severity of any infection, time of administration and other clinicalfactors. The dosage may vary with the type of matrix used in thereconstitution and with inclusion of other proteins in thepharmaceutical composition. For example, the addition of other knowngrowth factors, such as IGF I (insulin like growth factor I), to thefinal composition, may also effect the dosage. Progress can be monitoredby periodic assessment of tissue/bone growth and/or repair, for example,X-rays, histomorphometric determinations and tetracycline labeling.

Polynucleotides of the present invention can also be used for genetherapy. Such polynucleotides can be introduced either in vivo or exvivo into cells for expression in a mammalian subject. Polynucleotidesof the invention may also be administered by other known methods forintroduction of nucleic acid into a cell or organism (including, withoutlimitation, in the form of viral vectors or naked DNA).

Cells may also be cultured ex vivo in the presence of proteins of thepresent invention in order to proliferate or to produce a desired effecton or activity in such cells. Treated cells can then be introduced invivo for therapeutic purposes.

Patent and literature references cited herein are incorporated byreference as if fully set forth.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 5                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 374 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GCGGCCGCAGGTCTANAATTCAATCGGGCTGCCTTATCGCCAAGCTCCTTCAGGAGAACA60                AAGAACAGGCCATTACCCTGGAAAANACTGGCAACTGATTTTACCCACAAGCCCNAACCT120               CAGGGATTTCAGTATCTACTANTCTGGGTNNATACTTTCACGGGTTGGGCAGAAGCCTTC180               CCCTGTTGGACAGAAAANGCCCNAGANGTTNTAAAGGCACTAGTTCATGAAATAATTCCC240               ANATTCGGACTTCCCCGAGGCTTACAGANTGACNATNNCCCTGCTTTCCAGGCCACAGTN300               ACCCAGGGGAGTNTCCCNGGCGTTNGGTNTACGATATCACTTACACTGCGCCTGAANGCC360               ACAGTCCTCNGGGA374                                                             (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 542 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       AACAANTTNTTAAAACATTACAAGGAACGTATCCNTGAGNAGAGGGAAAAGAATTATTCC60                ACCCTTGTGACATGGTATTAGTCAAGTCCATTCCCTATAATTCCCNATCCCTAGANACAT120               CNTGGGANGGACCNTACCCAGTCATTTTATNTACCCCAACTGCGGTTAAAGTGGCTGGAG180               TGGAGTCTTGGATACATCACACTTGAGTCAAATCCTGGATACTGCCAAAGGAACCTGAAA240               ATCCAGGAGACAACGCTAGCTATTCCTGTGAACCTCTAGAGGATTTGCGCCTGCTCTTCA300               AACAACAACCAGGAGGAAAGTAACTAAAATCATAAATCCCCATGGCCCTCCCTTATCATA360               TTTTTCTCTTTACTGTTCTTTTACCCTCTTTCACTCTCACTGCACCCCCTCCATGCCGCT420               GTATGACCAGTAGCTCCCCTTACCAAGAGTTTCTATGGAGAATGCAGCGTCCCGGAAATA480               TTGATGCCCCATCGTATAGGAGTCTTTCTAAGGGAACCCCCACCTTCACTGCCCACACCC540               AT542                                                                         (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 67 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetAlaLeuProTyrHisIlePheLeuPheThrValLeuLeuProSer                              151015                                                                        PheThrLeuThrAlaProProProCysArgCysMetThrSerSerSer                              202530                                                                        ProTyrGlnGluPheLeuTrpArgMetGlnArgProGlyAsnIleAsp                              354045                                                                        AlaProSerTyrArgSerLeuSerLysGlyThrProThrPheThrAla                              505560                                                                        HisThrHis                                                                     65                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 279 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       RCCACATCCACCTTTAAACACGGGGNTTGCAAANAAGATNACACTTGACCAATCAGAGAG60                NTCANTAAAATGATNATTNGGCAAAAACAGGAGGTAAAGAAATAGCCAATCATCTATTGC120               CTGAGAGCACAGCAGGAGGGACAATGATCGGGATATAAACCCAAGTTTTNGAGCCGGCAA180               CGGCAACCCCCTTTGGGTCCCCTCCCTTTGTATGGGAGCTNTGTTTTCATGCTATTTCAN240               TNTATTAAATNTTGCAACTGCAAAAAAAAAAAAAAAAAA279                                    (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc ="oligonucleotide"                                     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CNTCCTGGTTGTTGTTTGAAGAGCAGGCG29                                               __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide selected from thegroup consisting of:(a) a polynucleotide comprising the nucleotidesequence of SEQ ID NO:2; (b) a polynucleotide comprising the nucleotidesequence of SEQ ID NO:2 from nucleotide 342 to nucleotide 542; (c) apolynucleotide comprising the nucleotide sequence of SEQ ID NO:2 fromnucleotide 402 to nucleotide 542; (d) a polynucleotide comprising thenucleotide sequence of the full length protein coding sequence of cloneAJ172₋₋ 2 deposited under accession number ATCC 98115; (e) apolynucleotide encoding the full length protein encoded by the cDNAinsert of clone AJ172₋₋ 2 deposited under accession number ATCC 98115;(f) a polynucleotide comprising the nucleotide sequence of the matureprotein coding sequence of clone AJ172₋₋ 2 deposited under accessionnumber ATCC 98115; (g) a polynucleotide encoding the mature proteinencoded by the cDNA insert of clone AJ172₋₋ 2 deposited under accessionnumber ATCC 98115; (h) a polynucleotide encoding a protein comprisingthe amino acid sequence of SEQ ID NO:3; (i) a polynucleotide encoding aprotein comprising a fragment of the amino acid sequence of SEQ ID NO:3from amino acid 21 to amino acid 67; and (j) a polynucleotide which isan allelic variant of a polynucleotide of (a)-(g) above.
 2. An isolatedpolynucleotide of claim 1 wherein said polynucleotide is operably linkedto an expression control sequence.
 3. A host cell transformed with apolynucleotide of claim
 2. 4. The host cell of claim 3, wherein saidcell is a mammalian cell.
 5. A process for producing a protein encodedby a polynucleotide of claim 2, which comprises:(a) growing a culture ofthe host cell of claim 3 in a suitable culture medium; and (b) purifyingsaid protein from the culture.
 6. A protein produced according to theprocess of claim
 5. 7. The protein of claim 6 comprising a matureprotein.
 8. An isolated protein, wherein said protein comprises an aminoacid sequence selected from the group consisting of:(a) the amino acidsequence of SEQ ID NO:3; (b) the amino acid sequence of SEQ ID NO:3 fromamino acid 21 to amino acid 67; and (c) the amino acid sequence encodedby the cDNA insert of clone AJ172₋₋ 2 deposited under accession numberATCC 98115;the protein being substantially free from other mammalianproteins.
 9. The protein of claim 8, wherein said protein comprises theamino acid sequence of SEQ ID NO:3.
 10. The protein of claim 8, whereinsaid protein comprises the amino acid sequence encoded by the cDNAinsert of clone AJ172₋₋ 2 deposited under accession number ATCC 98115.11. A composition comprising the protein of claim 8, and apharmaceutically acceptable carrier.
 12. The isolated gene correspondingto the cDNA sequence of SEQ ID NO:2, SEQ ID NO:1 and SEQ ID NO:4. 13.The polynucleotide of claim 1 wherein said polynucleotide comprises thenucleotide sequence of SEQ ID NO:2.
 14. The polynucleotide of claim 1wherein said polynucleotide comprises the nucleotide sequence of SEQ IDNO:2 from nucleotide 342 to nucleotide
 542. 15. The polynucleotide ofclaim 1 wherein said polynucleotide comprises the nucleotide sequence ofSEQ ID NO:2 from nucleotide 402 to nucleotide
 542. 16. Thepolynucleotide of claim 1 wherein said polynucleotide comprises thenucleotide sequence of the full length protein coding sequence of cloneAJ172₋₋ 2 deposited under accession number ATCC
 98115. 17. Thepolynucleotide of claim 1 wherein said polynucleotide comprises apolynucleotide encoding the full length protein encoded by the cDNAinsert of clone AJ172₋₋ 2 deposited under accession number ATCC 98115.18. The polynucleotide of claim 1 wherein said polynucleotide comprisesthe nucleotide sequence of the mature protein coding sequence of cloneAJ172₋₋ 2 deposited under accession number ATCC
 98115. 19. Thepolynucleotide of claim 1 wherein said polynucleotide comprises apolynucleotide encoding the mature protein encoded by the cDNA insert ofclone AJ172₋₋ 2 deposited under accession number ATCC
 98115. 20. Thepolynucleotide of claim 1 wherein said polynucleotide comprises apolynucleotide encoding a protein comprising the amino acid sequence ofSEQ ID NO:3.